PROJECT SUMMARY/ABSTRACT Membrane proteins are involved in many cellular processes and thus are critical drug targets for a wide range of diseases. However, there is a fundamental gap in understanding how the lipid environment affects membrane protein structure and function. Mounting evidence indicates that lipids can be essential for membrane protein function, but it is challenging to determine the molecular mechanisms underlying the importance of protein-lipid interactions. The primary challenge is that conventional structural biology tools and binding assays are poorly suited to characterizing transient and heterogeneous protein-lipid interactions. To understand how lipids modulate membrane protein structure and function, my research program is developing new tools to study protein-lipid interactions by combining lipoprotein nanodiscs with mass spectrometry (MS). These new technologies will be developed using well-characterized bacterial membrane protein complexes before being applied to more complex mammalian proteins such as rhodopsin and uncoupling protein 2. Our goal is to answer four questions for a given membrane protein target. 1) What lipids interact with the target? To identify the endogenous lipids that surround the target, we are developing a hybrid lipopeptide/lipoprotein approach to solubilize membranes surrounded by their natural lipids into nanodiscs without the need for detergent. Following purification of the target, we will extract and identify the lipids that are naturally associated with the target. 2) How strongly do lipids bind to the target? To distinguish tightly bound lipids from weakly associated lipids, we will assemble lipoprotein nanodiscs with different mixtures of lipids and use native MS to ionize the intact nanodisc assembly. Using collisional activation, we will gradually dissociate the nanodisc to measure the composition of the lipid annular belt and tightly associated structural lipids. Furthermore, we will use lipid exchange between nanodiscs to measure lipid binding constants in an experiment analogous to equilibrium dialysis. 3) Where do lipids interact with the protein structure? After predicting lipid- binding sites using molecular dynamics (MD), we will test the predictions by mutating interacting residues and using native MS to detect the disruption of the binding sites. 4) Why are specific protein-lipid interactions important for function? By pairing MD, mutagenesis, and native MS with functional studies, we will connect lipid-dependent effects on protein function with specific lipid binding sites. Our overarching goal is to develop a toolbox for unravelling the molecular mechanisms of protein-lipid interactions. This will impact biomedical research by identifying lipids important for maintaining protein activity and aiding in elucidating the physiological mechanisms of membrane proteins inside natural bilayers. Ultimately, an improved understanding of protein-lipid interactions holds the potential for improved drug discovery with membrane protein targets and for new therapeutic strategies for modulating membrane protein activity.